JP2022088645A - Treatment of hyperbilirubinemia - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a nucleic acid sequence useful in the treatment of Crigler-Najjar syndrome, an autosomal recessive disorder with severe unconjugated hyperbilirubinemia due to deficiency of bilirubin UDP-glucuronosyltransferase isozyme 1A1 (UGT1A1).

SOLUTION: The present invention discloses a nucleic acid sequence which is a codon optimized UGT1A1 coding sequence, wherein the optimized coding sequence has an increased GC content and/or has a decreased number of alternative open reading frames as compared to the wild-type coding sequence.

SELECTED DRAWING: None

COPYRIGHT: (C)2022,JPO&INPIT

Description

INDUSTRIAL APPLICABILITY The present invention relates to nucleic acid sequences useful in the treatment of hyperbilirubinemia, particularly Crigler-Najr syndrome. More specifically, the nucleic acid sequence of the invention is a codon-optimized human UGT1A1 coding sequence.

Background of the Invention Crigler-Najer syndrome (CN) has severe unconjugated hyperbilirubinemia due to a deficiency of the bilirubin UDP-glucuronosyltransferase isozyme 1A1 (UGT1A1) encoded by the UGT1A1 gene (OMIM218800). It is an autosomal recessive disorder. The prevalence of CN is about 1 in 1,000,000 at birth, which makes CN an extremely rare disease. Current therapy for CN relies on phototherapy to prevent elevated serum bilirubin levels. In a mild form, also known as CNII type, phenobarbital can be used to reduce bilirubinemia. Nevertheless, patients are potentially at risk of high life-threatening levels of bilirubin in the blood, and liver transplantation remains the only curative treatment. In its most severe form, if phototherapy is not applied from birth, the disease is fatal due to bilirubin-induced neuropathy. Despite the availability of therapy, CN still has diminished efficacy of growing phototherapy, poor compliance due to the limitations of phototherapy itself (must be done 10-12 hours daily), and Medical needs are not met for a number of reasons, including the development of pathological liver changes over time, which may require liver transplantation.

A more recent knock-in mouse model of the disease (Bortolussi et al., 2012) developed by Dr. Muro of ICGEB in Trieste, Italy, which has the same mutations found in naturally occurring Gunn rats and Gunn rats. There are various animal models of the disease, including. Gunn rats exhibit high bilirubin levels in serum and they have cerebellar hypoplasia; CN mice have a much more severe phenotype and soon after birth unless treated rapidly with phototherapy or gene therapy. Died in (Bortolussi et al., 2012).

Previous studies aimed at developing gene-based therapies for CN have shown that therapeutic efficacy can be achieved using AAV vectors delivered to the liver (Bortolussi et al., 2012; Seppen et al., 2006). However, there is still a need for more effective treatment strategies.

Gilbert's syndrome (ie, GS; OMIM218800) is a hereditary liver disease and is the most common cause of hereditary bilirubin hyperplasia. It is found in less than 3-12% of the population. GS is also caused by mutations in the UGT1A1 gene. Therefore, therapeutic strategies aimed at reducing hyperbilirubinemia will also be favorably implemented in the treatment of GS.

Description of the Invention The present invention relates to an optimized UGT1A1 coding sequence of codons derived from the human UGT1A1 cDNA. More specifically, the codon-optimized UGT1A1 coding sequence has an increased GC content and / or a reduced number of alternative open reading frames compared to the wild-type human coding sequence of SEQ ID NO: 1. .. For example, the nucleic acid sequence of the present invention has an increased GC content of the UGT1A1 sequence by at least 2, 3, 4, 5, or 10% as compared to the sequence of the wild-type human UGT1A1 sequence. In certain embodiments, the nucleic acid sequences of the invention have a GC content of the UGT1A1 sequence of 2, 3, 4, or more preferably 5% or 10% (preferably 5% or 10%) as compared to the sequence of the wild-type human UGT1A1 sequence. 5%) It is increasing. In certain embodiments, the nucleic acid sequences of the invention encoding the codon-optimized human UGT1A1 protein are "substantially identical", i.e., relative to the sequence of SEQ ID NO: 2 or SEQ ID NO: 3. About 70% identical, more preferably about 80% identical, even more preferably about 90% identical, even more preferably about 95% identical, even more preferably about 97%, 98%, or even 99% identical. .. In certain embodiments, the present invention relates to a nucleic acid sequence encoding a codon-optimized human UGT1A1 protein, wherein the nucleic acid sequence comprises the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3.

Advantageously, the codon-optimized nucleic acids of the invention provide improved and / or reduced immunogenicity of bilirubin levels.

The present invention also relates to nucleic acid constructs comprising the nucleic acid sequences of the present invention. Nucleic acid constructs can correspond to expression cassettes containing the nucleic acid sequences of the invention that are operably linked to one or more expression control sequences, or other sequences that enhance expression of the transgene. Such sequences, such as promoters, enhancers, introns, poly A signals, etc., are known in the art. In particular, the expression cassette may include a promoter. The promoter can be a ubiquitous or tissue-specific promoter, particularly a liver-specific promoter. More specifically, the promoters include liver-specific promoters such as the α-1 antitrypsin promoter (hAAT) (SEQ ID NO: 4), transthyretin promoter, albumin promoter, thyroxine-binding globulin (TBG) promoter and the like. Other useful liver-specific promoters, such as those listed in the Cold Spring Harbor Laboratory-edited liver-specific gene promoter database (http://rulai.cshl.edu/LSPD/), are in the art. It is known. Representative ubiquitous promoters include cytomegalovirus enhancer / chicken β-actin (CAG) promoter, cytomegalovirus enhancer / promoter (CMV), PGK promoter, SV40 initial promoter and the like. In certain embodiments, the promoter is set forth in an ApoE regulatory region, eg, a human ApoE regulatory region (or human apolipoprotein E / C-I locus, liver regulatory region HCR-1-Genbank accession number U32510, SEQ ID NO: 11). It is bound to an enhancer sequence such as). In certain embodiments, the enhancer sequence, eg, the ApoE sequence, is bound to the promoters listed above, particularly liver-specific promoters such as the hAAT promoter.

In certain embodiments, the nucleic acid construct comprises an intron, in particular an intron located between the promoter and the coding sequence. Introns may be introduced to enhance mRNA stability and protein production. In certain embodiments, the nucleic acid construct comprises a human β-globin b2 (ie, HBB2) intron, a coagulation factor IX (FIX) intron, an SV40 intron, or a chicken β-globin intron. In certain embodiments, the nucleic acid constructs of the invention are modified introns designed to reduce or even completely eliminate the number of alternative open reading frames (ARFs) found in the intron. It contains a particularly modified HBB2 or FIX intron). Preferably, the ARF, whose length extends over 50 bp and has a stop codon within the frame with the start codon, is removed. ARF can be removed by modifying the intron sequence. For example, modifications can be made by nucleotide substitutions, insertions, or deletions, preferably nucleotide substitutions. As an example, substituting one or more nucleotides, in particular one nucleotide, in the start codon of ATG or GTG present in the sequence of the intron of interest can result in a codon that is not the start codon. For example, the ATG or GTG in the intron sequence of interest can be replaced by a CTG that is not the start codon.

The classic HBB2 intron used for nucleic acid constructs is shown in SEQ ID NO: 5. For example, this HBB2 intron can be modified by eliminating the start codons (ATG and GTG codons) within the intron. In certain embodiments, the modified HBB2 intron contained in the construct has the sequence set forth in SEQ ID NO: 6. The classic FIX intron used for nucleic acid constructs is derived from the first intron of human FIX, which is shown in SEQ ID NO: 7. The FIX intron can be modified by eliminating the start codons (ATG and GTG codons) within the intron. In certain embodiments, the modified FIX intron contained in the construct of the invention has the sequence set forth in SEQ ID NO: 8. The classical chicken β-globin intron used in nucleic acid constructs is shown in SEQ ID NO: 9. The chicken-β-globin intron can be modified by eliminating the start codons (ATG and GTG codons) within the intron. In certain embodiments, the modified chicken-β-globin intron contained in the construct of the invention has the sequence set forth in SEQ ID NO: 10.

We have shown that such modified introns, especially modified HBB2 or FIX introns, have advantageous properties and can significantly improve the expression of the transgene. Furthermore, it is believed that by reducing the number of ARFs in the intron contained within the construct of the present invention, the immunogenicity of the construct is also reduced.

Accordingly, the present invention also relates to an intron, which is intended to be used in an expression cassette and has been modified to increase the expression efficiency of the transgene located in the cassette. In particular, the present invention relates to modified introns derived from known introns, where the number of ARFs has been reduced or the ARFs have been completely eliminated. In certain embodiments, the present invention relates to a modified HBB2 intron having a reduced number of ARFs or no ARFs at all. In a further specific embodiment, the modified HBB2 intron is the intron set forth in SEQ ID NO: 6. In another embodiment, the invention relates to a modified FIX intron having a reduced number of ARFs or no ARFs at all. In a further specific embodiment, the modified FIX intron is the intron set forth in SEQ ID NO: 8. In another embodiment, the invention relates to a modified chicken β-globin intron having a reduced number of ARFs or no ARFs at all. In a further specific embodiment, the modified chicken β-globin intron is the intron set forth in SEQ ID NO: 10. A further aspect of the invention relates to nucleic acid constructs, vectors, such as viral vectors, particularly AAV vectors, and cells containing the modified introns of the invention. Nucleic acid constructs may include additional expression control sequences, such as promoters and / or enhancers, such as those described herein. Modified introns as disclosed herein enhance the expression efficiency of transgenes placed in nucleic acid constructs, eg, therapeutic genes. In the context of this aspect of the invention, "therapeutic gene" generally refers to a gene encoding a therapeutic protein that is useful in treating a medical condition. When expressed, a therapeutic gene imparts a beneficial effect on the cell or tissue in which it is present, or on the patient in which the gene is expressed. Examples of beneficial effects include remission of signs or symptoms of condition or disease, prevention or suppression of condition or disease, or impartation of desired characteristics. Therapeutic genes include genes that partially or completely correct a patient's gene defect. In particular, therapeutic genes are useful in gene therapy to alleviate deficiencies caused by the protein levels that are defective, incomplete, or suboptimal in the subject's cells or tissues. It can be, but is not limited to, a nucleic acid sequence encoding a protein. Therefore, the invention comprises nucleic acid constructs, vectors such as viral vectors, particularly AAV vectors, which include the modified introns of the invention and also contain therapeutic genes of interest for use in gene therapy. Regarding cells. The present invention can generally be applied to the treatment of any disease that can be treated by the expression of a therapeutic gene in a subject's cells or tissues. These include, for example, proliferative diseases (cancer, tumors, dysplasia, etc.), infectious diseases; viral diseases (eg, induced by hepatitis B or C virus, HIV, herpes, retrovirus, etc.); hereditary. Diseases (cystic fibrosis, dystroglycanopathies, myopathy, eg Duchenne-type myopathy; myotube myopathy; hemophilia; sacral erythrocyte anemia, sickle erythema, fancony anemia; diabetes; muscular atrophic lateral sclerosis Disease, mononeurones disease, such as spinal muscle atrophy, bulbar spinal muscle atrophy, or Sharko Marie Tooth's disease; arthritis; severe complex immunodeficiency (eg RS-SCID, ADA-SCID, or X-SCID), Wiscot-Aldrich syndrome, X-chain thrombocytopenia, X-chain congenital neutrophilia, chronic granulomatosis, etc.), cardiovascular disease (re-stenosis, ischemia, dyslipidemia, homozygosity) Sexual familial hypercholesterolemia, etc.) or neurological disorders (psychiatric disorders, neurodegenerative disorders such as Parkinson's disease or Alzheimer's disease, Huntington's disease, indulgence (eg for tobacco, alcohol, or drugs), epilepsy, canavan's disease, adrenal White dystrophy, etc.), eye diseases such as retinal pigment degeneration, Labor congenital melanosis, Labor hereditary optic neuropathy, Stargart's disease; lysosome accumulation disease, such as Sanfilipo syndrome; hyperbilylbinemia, such as CNI or II, or Gilbert syndrome, Pompe disease and the like. In order to successfully express a transgene, such as a therapeutic gene, in a recipient host cell, it is preferably a promoter, its own promoter or heterologous, as described above and further developed in the disclosure below. It is operably linked to one of the promoters of. Many suitable promoters are known in the art and the choice is the desired expression level of the product encoded by the therapeutic gene; whether constitutive expression, cell-specific expression, or tissue-specific expression is desired. It depends on whether or not. The nucleic acid construct containing the modified intron, the vector containing the nucleic acid construct, or the construct or the cell containing the vector is further if the gene of interest is a therapeutic gene as defined above. , Can be used for gene therapy or cell therapy.

In certain embodiments, the nucleic acid constructs of the invention are promoters optionally placed in front of enhancers in the 5'to 3'direction, optimized UGT1A1 coding sequences of codons of the invention, and polyadenylation signals. It is an expression cassette containing. In certain embodiments, the nucleic acid constructs of the invention are promoters, introns (particularly as defined above) that are optionally placed in front of enhancers (eg, ApoE control regions) in the 5'to 3'direction. An expression cassette containing an intron), an optimized UGT1A1 coding sequence for the codons of the invention, and a polyadenylation signal. In a further specific embodiment, the nucleic acid constructs of the invention are enhancers, eg, ApoE control regions, promoters, introns (particularly introns as defined above), codons of the invention in the 5'to 3'direction. An expression cassette containing an optimized UGT1A1 coding sequence and a polyadenylation signal.

The invention also relates to vectors containing nucleic acid sequences as disclosed herein. In particular, the vector of the present invention is a vector suitable for use in gene therapy. For example, the vector can be a plasmid vector. More specifically, the vector is a viral vector suitable for gene therapy targeting liver tissue or hepatocytes. In this case, the nucleic acid constructs of the invention also contain sequences suitable for producing effective viral vectors, as is well known in the art. In a more specific embodiment, the viral vector is an AAV vector, eg, an AAV vector suitable for transfecting liver tissue or hepatocytes, more specifically AAV-1, -2, -5, -6, -7, Vectors such as -8, -9, -rh10, -rh74, -dj or retroviral vectors such as lentiviral vectors. In a further embodiment, the AAV vector comprises a genome that is either single-stranded or self-complementary double-stranded. Preferably, for the practice of the present invention, the AAV genome is single-stranded. As is known in the art, appropriate sequences will be introduced into the nucleic acid constructs of the invention in order to obtain a functional viral vector, depending on the specific viral vector conceivable for the application. .. Suitable sequences include AAV ITR for AAV vectors or LTR for lentiviral vectors. Accordingly, the invention also relates to an expression cassette as described above, flanked by the ITR or LTR on each side.

In a particularly preferred embodiment, the invention relates to an AAV vector comprising a nucleic acid construct of the invention in a single-stranded or double-stranded self-complementary genome (eg, a single-stranded genome). In certain embodiments, the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3. In one embodiment, the AAV vector is an AAV8 vector. In a further specific embodiment, the nucleic acid is operably linked to a promoter, particularly a ubiquitous or liver-specific promoter. According to certain variants, the promoters are ubiquitous promoters such as cytomegalovirus enhancer / chicken β-actin (CAG) promoter, cytomegalovirus enhancer / promoter (CMV), PGK promoter, and SV40 early promoter. In certain variants, the ubiquitous promoter is the CAG promoter. According to another variant, the promoters are liver-specific promoters such as the α-1 antitrypsin promoter (hAAT), transthyretin promoter, albumin promoter, and thyroxine-binding globulin (TBG) promoter. In a particular variant, the liver-specific promoter is the hAAT liver-specific promoter of SEQ ID NO: 4. In a more specific embodiment, the nucleic acid construct included in the genome of the AAV vector of the invention further comprises an intron as described above, eg, an intron located between a promoter and a nucleic acid sequence encoding the UGT1A1 protein. include. Representative introns that can be included in nucleic acid constructs introduced into the AAV vector genome include, but are not limited to, human β-globin b2 (ie, HBB2) introns, FIX introns, and chicken β-globin introns. The intron in the genome of the AAV vector may be a classical (ie, unmodified) intron, or the number of alternative open reading frames (ARFs) within the intron may be reduced or even completely. It may be a modified intron designed to be removed. Modified and unmodified introns that can be used in the practice of this embodiment in which the nucleic acids of the invention are introduced into the AAV vector are fully described above. In certain embodiments, the AAV vector of the invention, in particular the AAV8 vector, has a modified (ie, optimized) intron in its genome, eg, a modified HBB2 intron of SEQ ID NO: 7, a modification of SEQ ID NO: 8. Includes FIX introns, and modified chicken β-globin introns of SEQ ID NO: 10.

The invention also relates to cells, such as hepatocytes, transformed with the nucleic acid sequences of the invention. The cells of the invention may be delivered to a subject in need thereof via injection into the liver or bloodstream of the subject. In certain embodiments, the invention introduces the nucleic acid sequence of the invention into hepatocytes, particularly into the hepatocytes of the subject to be treated, and administers the nucleic acid-introduced hepatocytes to the subject. including.

The invention also provides a pharmaceutical composition comprising a nucleic acid of the invention, a vector of the invention, or an active substance selected from the cells of the invention in combination with a pharmaceutically acceptable carrier.

The present invention also relates to a method for treating hyperbilirubinemia caused by a mutation in the UGT1A1 gene, which comprises the step of delivering the nucleic acid, vector, pharmaceutical composition, or cell of the present invention to a subject in need thereof. .. In certain embodiments, hyperbilirubinemia is type CNI or type II, or Gilbert's syndrome.

The invention also relates to the nucleic acids, vectors, pharmaceutical compositions, or cells of the invention for use as pharmaceuticals.

The invention also presents the nucleic acids, vectors, pharmaceuticals of the invention for use in the treatment of hyperbilirubinemia caused by mutations in the UGT1A1 gene, particularly CNI or type II, or Gilbert's syndrome. It also relates to compositions or cells.

The present invention further relates to the nucleic acids, vectors, pharmaceuticals of the present invention in the manufacture of pharmaceuticals useful for the treatment of hyperbilirubinemia caused by mutations in the UGT1A1 gene, particularly for the treatment of CNI type or type II, or Gilbert's syndrome. Also related to the use of compositions or cells.

Detailed Description of the Invention The term "UGT1A1" refers to the wild-type homosapiens UDP-glucuronosyltransferase 1 family 1, polypeptide A, (UGT1A1) cCDNA set forth in SEQ ID NO: 1 (Axon No. NM_000463.2, This is a reference sequence for CDS of UGT1A1 human mRNA; reference number 191740 in OMIM).

The term "codon-optimized" is a codon that expresses a bias towards humans (ie, common in human genes, but not in other mammalian or non-mammalian genes. ) Means that it has changed to a synonymous codon (a codon that encodes the same amino acid) that does not express a bias toward humans. Therefore, changes in codons do not result in any amino acid changes in the encoded protein.

The sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3, particularly the sequence set forth in SEQ ID NO: 2, is a preferred embodiment of the codon-optimized nucleic acid sequence of the present invention.

Changes in the DNA sequences derived from codon optimization in SEQ ID NO: 2 and SEQ ID NO: 3 result in an increase in GC content of the UGT1A1 sequence by about 5% and about 10%, respectively.

Also, they are "substantially identical", i.e., about 70% identical, more preferably about 80% identical, even more preferably about 90% identical, even more preferred to the sequence of SEQ ID NO: 2 or SEQ ID NO: 3. Also included by the invention are the nucleic acid sequences of the invention encoding a codon-optimized human UGT1A1 protein that are about 95% identical, even more preferably about 97%, 98%, or even 99% identical. Will be done.

"Identity" refers to sequence identity between two nucleic acid molecules. If the position in both of the two compared sequences is occupied by the same base, for example if the position in each of the two DNA molecules is occupied by adenine, the molecule is identical at that position. The same ratio between two sequences is a function of dividing the number of matched positions shared by the two sequences by the number of positions to be compared and multiplying it by 100. For example, if 6 of the 10 positions match in 2 sequences, then the 2 sequences are 60% identical. Generally, the two sequences are aligned and compared so that the maximum identity is obtained. Nucleic acid sequences can be aligned using various bioinformatics tools known to those of skill in the art, such as BLAST or FASTA.

The term "reduced immunogenicity" as applied to a codon-optimized UGT1A1 coding sequence or a modified intron of the invention refers to the codon-optimized gene or modified intron. It contains a reduced number of potential alternative open reading frames (ie, ARFs) in the intron, and / or in the coding sequence, thereby particularly compared to wild-type cDNA or other UGT1A1 cDNA variants. This means limiting the number of potential translation protein by-products from the encoded mRNA. In particular, ARFs having a stop codon within a frame having a length of 50 bp or more and having a start codon are reduced.

In the context of the present invention, the term "gene therapy" refers to the treatment of a subject, including delivery of the gene / nucleic acid to individual cells for the purpose of treating the disease. Gene delivery is generally accomplished using a delivery vehicle, also known as a vector. Viral and non-viral vectors can be used to deliver the gene to the patient's cells. Particularly preferred are AAV vectors, especially AAV8 vectors.

It will be appreciated that the nucleic acids of the invention may contain one or more polyadenylation signals, typically located at the 3'end of the molecule.

Preferred vectors for delivering the nucleic acids of the invention are viral vectors such as retroviral vectors such as lentiviral vectors or non-pathogenic parvoviruses, more preferably AAV vectors. Adeno-associated virus (AAV) of human parvovirus is a naturally replication-deficient dependent virus that can be integrated into the genome of infected cells to establish latent infection. The last property appears to be unique among mammalian viruses. This is because integration occurs at a specific site in the human genome called AAVS1 located on chromosome 19 (19q13.3-qter).

Therefore, AAV has received considerable interest as a potential vector for human gene therapy. Preferred properties of the virus are that it is completely free of human disease, that it can infect both dividing and non-dividing cells, and that it can infect diverse cell lines from different tissues.

Among the well-characterized AAV serotypes isolated from human or non-human primates (NHP), human serotype 2 is the first AAV developed as a gene transfer vector. Other currently used AAV serotypes include AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAVrh74, and AAVdj. In addition, non-naturally engineered mutants and chimeric AAVs may also be useful.

AAV viruses can be engineered using conventional molecular biological techniques and these particles are stable to deliver cell-specific nucleic acid sequences and to minimize immunogenicity. And to adjust the lifetime of the particles, for effective degradation, it can be optimized for accurate delivery to the nucleus.

Desirable AAV fragments for construction into vectors encode cap proteins (including vp1, vp2, vp3 and hypervariable regions), rep proteins (including rep78, rep68, rep52, and rep40), and these proteins. Contains an array. These fragments can be readily utilized in a variety of vector systems and host cells.

Rep protein-deficient AAV-based recombinant vectors exist primarily as stable cyclic episomes that integrate into the host's genome with low efficiency and can persist for several years in target cells.

Instead of using native AAV serotypes, artificial AAV serotypes (eg, including but not limited to AAV with non-natural capsid proteins) can be used in the context of the invention. Such artificial capsids are obtained by any suitable technique from different selected AAV serotypes, from discontinuous portions of the same AAV serotype, from non-AAV viral sources, or from non-viral sources. It can be made using a selected AAV sequence (eg, a fragment of the vp1 capsid protein) in combination with a possible heterologous sequence. Artificial AAV serotypes can be, but are not limited to, chimeric AAV capsids, recombinant AAV capsids, or "humanized" AAV capsids.

Accordingly, the invention relates to an AAV vector comprising the nucleic acid of the invention, which is a codon-optimized UGT1A1 coding sequence. In the context of the present invention, AAV vectors include AAV capsids capable of transducing target cells of interest, especially hepatocytes. According to certain embodiments, the AAV vector is a serotype such as AAV-1, -2, -5, -6, -7, -8, -9, -rh10, -rh74, -dj. In a more specific embodiment, the AAV vector is a pseudotyped vector, i.e. its genome and capsids are derived from AAVs of different serotypes. For example, a pseudotyped AAV vector has its genome derived from AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh10, rh74, or dj serotype, and its capsid is another serum. It can be a serotype-derived vector. For example, the genome of the pseudotyped vector can be derived from the AAV1, 2, 3, 4, 5, 6, 7, 10, rh10, rh74, or dj serotype, the capsid of which is derived from the AAV8 or AAV9 serotype. , Especially derived from the AAV8 serotype.

In another embodiment, the capsid is a modified capsid. In the context of the present invention, a "modified capsid" can be a chimeric capsid or a capsid containing one or more mutant VP capsid proteins derived from one or more wild-type AAV VP capsid proteins. .. In certain embodiments, the AAV vector is a chimeric vector, i.e., the capsid comprises a VP capsid protein from at least two different AAV serum types, or a VP protein from at least two AAV serum types. Includes at least one chimeric VP protein with regions or domains coalesced. Examples of such chimeric AAV vectors useful for transducing hepatocytes are described in Shen et al., Molecular Therapy, 2007 and Tenney et al., Virology, 2014. For example, a chimeric AAV vector can be obtained from a combination of an AAV8 capsid sequence with a sequence of AAV1, 2, 3, 4, 5, 6, 7, 9, 10, rh10, rh74, or dj serotype. In another embodiment, the capsid of the AAV vector is one or more mutant VP capsid proteins that exhibit high directivity to the liver, eg, those described in WO 20150131313, in particular RHM4-1, RHM15-1. , RHM15-2, RHM15-3 / RHM15-5, RHM15-4 and RHM15-6 capsid variants.

In another embodiment, the modified capsid is also obtained from the modification of the capsid inserted by error-prone PCR and / or peptide insertion (eg, described in Bartel et al., 2011). In addition, capsid variants may contain single amino acid changes such as tyrosine mutants (see, eg, Zhong et al., 2008).

In addition, the genome of the AAV vector can be either a single-stranded or self-complementary double-stranded genome (McCarty et al., Gene Therapy, 2003). Self-complementary double-stranded AAV vectors are made by deleting terminal resolution sites (trs) from one of the AAV terminal repeats. These modified vectors, whose replication genome is half the length of the wild-type AAV genome, tend to package DNA dimers. In a preferred embodiment, the AAV vector performed in practice of the invention has a single-stranded genome, more preferably AAV8, AAV2, or AAV5 capsid, more preferably AAV8 capsid.

Apart from the specific delivery systems embodied below in the Examples, various delivery systems are known and can be used to administer the nucleic acids of the invention, eg, liposomes, microparticles, micros. Encapsulation, encapsulation into recombinant cells capable of expressing an optimized UGT1A1 coding sequence for codons, receptor-mediated endocytosis, construction of therapeutic nucleic acids as part of retroviruses or other vectors, etc. .. Nucleic acid administration methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The nucleic acid sequences of the invention, whether vectorized or not, can be lined with epithelial or mucocutaneous mucosa (eg, oral mucosa, rectal mucosa, and intestinal mucosa, etc.) by any conventional route, eg, by injection or bolus injection. Can be administered by absorption through) and can be administered with other biologically active substances. Administration can be systemic or topical. Furthermore, it may be desirable to introduce the pharmaceutical composition of the present invention into the liver of a subject by any suitable route. In addition, bare DNA, such as minicircles and transposons, can be used for delivery or lentiviral vectors. In addition, gene editing techniques such as zinc finger nucleases, meganucleases, TALENs, and CRISPRs can also be used to deliver the coding sequences of the invention.

In certain embodiments, it may be desirable to administer the pharmaceutical composition of the invention topically to the area of need for treatment, i.e. the liver. This can be achieved, for example, with an implant, which is a porous, non-porous, or gel material, such as a membrane, such as a membrane made of silicon, or a fiber.

In another embodiment, the nucleic acids of the invention can be delivered in vesicles, especially liposomes.

In yet another embodiment, the nucleic acids of the invention can be delivered in a release control system.

The present invention also provides a pharmaceutical composition comprising the nucleic acid of the invention, or the vector of the invention, or the cell of the invention. Such compositions include therapeutically effective amounts of therapeutic agents (nucleic acids, vectors, or cells of the invention) and pharmaceutically acceptable carriers. In certain embodiments, the term "pharmaceutically acceptable" is approved by federal or state government regulators, or for use in the United States Pharmacopeia or the European Pharmacopoeia, or in animals and humans. Means listed in other commonly accepted pharmacopoeias for. The term "carrier" refers to a diluent, adjunct, excipient, or vehicle to which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids such as water and oils such as crude oils, animals, plants, or oils of synthetic origin such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is the preferred carrier for intravenous administration of the pharmaceutical composition. Aqueous saline and aqueous dextrose and glycerol solutions can also be used as liquid carriers, especially for injections. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol and the like.

The composition may also contain a small amount of wetting or emulsifying agent, or pH buffering agent, if desired. These compositions can be in the form of liquids, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Oral formulations may include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such a composition will contain a therapeutically effective amount of therapeutic agent, preferably in purified form, along with an appropriate amount of carrier to provide a dosage form for appropriate administration to the subject. .. In certain embodiments, the nucleic acids, vectors, or cells of the invention are formulated in a composition comprising phosphate buffered saline and supplemented with 0.25% human serum albumin. In another particular embodiment, the nucleic acid, vector, or cell of the invention is lactic acid at a final concentration of 0.01-0.0001% by weight, eg 0.001% by weight, based on the weight of the total composition. It is formulated in a composition comprising Ringer's solution and a nonionic surfactant, such as Pluronic F68. The preparation may further contain serum albumin, in particular human serum albumin, such as 0.25% human serum albumin. Other suitable formulations for either storage or administration are known in the art, in particular from WO 2005/118792 or Allay et al., 2011.

In a preferred embodiment, the composition is formulated according to the same conventional procedure as a pharmaceutical composition suitable for intravenous administration to humans. Typically, the composition for intravenous administration is a solution in sterile isotonic aqueous buffer. If desired, the composition may also include a solubilizer and a local anesthetic to relieve pain at the injection site, such as lignokine.

The amount of therapeutic agent of the invention (ie, nucleic acid, vector, or cell) that may be effective in treating CN can be determined by standard clinical techniques. In addition, in vivo and / or in vitro assays can optionally be used to help predict optimal dose ranges. The exact dose used in the formulation also depends on the route of administration and the severity of the disease and should be determined according to the practitioner's judgment and the circumstances of each patient. The dose of nucleic acid, vector, or cell administered to the subject in need includes the route of administration, the specific disease to be treated, the age of the subject, or the level of expression required for the required therapeutic effect. It will change based on several factors, not limited. One of ordinary skill in the art can readily determine the range of dosages required based on these and other factors, based on his knowledge in the art. For procedures involving administration of a viral vector, eg, an AAV vector, to a subject, a typical vector dose is at least 1 × 10 ⁸ vector genomes (vg / kg) per kg body weight, eg, at least 1 × 10. ⁹ vg / kg, at least 1 x 10 ¹⁰ vg / kg, at least 1 x 10 ¹¹ vg / kg, at least 1 x 10 ¹² vg / kg, at least 1 x 10 ¹³ vg / kg, or at least 1 x 10 ¹⁴ vg / kg Is.

FIG. 1 shows the levels of messenger RNA observed in Huh-7 cells transfected with a plasmid expressing wild-type UGT1A1 or an optimized UGT1A1 sequence of two codons (panel A), and. Includes a graph showing Western blot quantification (panel B) of UGT1A1 protein in the same sample. FIG. 2 includes a graph showing the effect of various intron optimizations on luciferase expression (panel A) and the effect of HBB2 optimization on RNA and protein expression levels of UGT1A1 (panel B). FIG. 3 shows two vectors containing an optimized UGT1A1 coding sequence for codons and containing either wild-type (UGT1A1 2.0) or optimized (UGT1A1 2.1) HBB2 introns. It is a photograph of a Western blot gel showing the expression of the UGT1A1 protein from. FIG. 4 is a computerized illustration of an alternative reading frame (ARF) within a wild-type UGT1A1 vector (A) and a codon-optimized UGT1A1 v2.1 vector (B). FIG. 5 is a graph showing total bilirubin (TB) levels measured weekly after injection of codon-optimized UGT1A1 vector or PBS into various rat strains. FIG. 6 shows total bilirubin (TB) levels measured weekly after injection of low dose codon-optimized UGT1A1 vector or PBS into various rat strains (compared to the data reported in FIG. 5). ) Is shown in the graph. FIG. 7 is a graph showing (A) total bilirubin (TB) levels measured weekly after injection of three UGT1A1 vectors (compared to the data reported in FIG. 8); (B) three vectors. Western blot photographs of liver extracts obtained from rats treated with and their relative quantification; and (C) AAV8-v2.1 4 months after injection into both male and female animals. Includes a graph presenting a long-term assessment of the efficacy of UGT1A1. FIG. 8 is a graph showing the ability of various constructs to correct severe hyperbilirubinemia (total bilirubin, expressed as mg / dl) in a mouse model of Crigler-Najer syndrome. Untreated animals (UNTR) have been reported.

Scope: Throughout this disclosure, various aspects of the invention can be presented in the form of a range. It should be understood that the description in the form of a range is solely for convenience and conciseness and should not be considered as an inflexible limitation on the scope of the invention. Therefore, the description of a range should be considered to have all possible subranges specifically disclosed, as well as individual numerical values within that range. For example, the description of the range such as 1 to 6 is the partially disclosed partial range such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and the range thereof. It should be considered to have individual numbers within, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of range.

The disclosure of each and each patent, patent application, and publication cited herein is thereby incorporated herein by reference in its entirety.

Without further description, one of ordinary skill in the art would be able to manufacture and use the compounds of the invention and practice the claimed method using the above description and the following exemplary examples. ..

Examples The present invention is further detailed by reference to the following experimental examples and accompanying drawings. These examples are provided for illustration purposes only and are not intended to limit them.

Materials and Methods Codon Optimization and AAV Vector Constructs:
UGT1A1 has undergone codon optimization according to several different algorithms. In addition, removal of the hidden transcription initiation site was performed on the entire construct. The resulting construct was either introduced into an expression plasmid or packaged in an AAV serotype 8 vector and tested in vitro and in vivo (rats and mice) for efficacy.

The following abbreviations for these constructs are used throughout this experimental unit:
-WT. 0: Wild-type UGT1A1 transgene and wild-type HBB2 intron (SEQ ID NO: 5);
-WT: Optimized version of the HBB2 intron (SEQ ID NO: 6) with the wild-type UGT1A1 transgene and some ARF removed;
-V2 (or v2.0): Includes codon-optimized UGT1A1 transgene version 2.0 (SEQ ID NO: 2) and wild-type HBB2 intron (SEQ ID NO: 5);
-V2.1: Includes an optimized version of the codon-optimized UGT1A1 transgene version 2.0 (SEQ ID NO: 2) and an optimized version of the HBB2 intron (SEQ ID NO: 6) with some ARF removed;
-V3: Codon-optimized UGT1A1 transgene version 3 (SEQ ID NO: 3) is included with an optimized version of the HBB2 intron (SEQ ID NO: 6) with some ARF removed;
-AAV8-hAAT-wtUGT1A1: AAV8 vector containing wild-type constructs under the control of the hAAT promoter wild-type UGT1A1 transgene;
-AAV8-hAAT-coUGT1A1v2: AAV8 promoter containing the v2 construct under the control of the hAAT promoter;
-AAV8-hAAT-coUGT1A1v2.1: AAV8 vector containing the v2.1 construct under the control of the hAAT promoter;
-AAV8-hAAT-coUGT1A1v3: AAV8 vector containing the v3 construct under the control of the hAAT promoter wild type.

In vitro assay:
The human hepatocyte cell line Huh7 was transduced with an increasing multiplicity of infection (MOI) of 0, 5000 (5), 10,000 (10), or 25,000 (25), or the designated plasmid and lipofectamine. Transfected using. Forty-eight hours after transduction, cells were collected, lysed, microsome extracts were prepared and placed on Western blots, where proteins were detected using polyclonal antibodies against human UGT1. The constitutively expressed protein calnexin was used as a loading control.

The portion of the cell used for microsome preparation was used for the extraction of mRNA with trizol. The extracted mRNA was treated with DNAse, reverse transcribed, and analyzed by RT-PCR using oligonucleotide primers specific for the UGT1A1 sequence. Oligonucleotide primers specific for human serum alkaline phosphatase were used for standardization.

Computer analysis:
Alternative Reading Frame (ARF) analysis was performed on the coding strands of the two UGT1A1 sequences using the ORF analysis tool present in the VectorNTI software (Life Technologies) Classic start, utilizing termination sites for eukaryotic cells. (ATG as the starting site and TAA TGA TAG as the ending site, respectively). ARF was considered when its length was greater than or equal to 50 bp and it had a stop codon within the frame with the start codon.

animal:
Vectors were injected into 6-8 week old Gunn rats exhibiting a defect in the UGT1A1 gene. The vector was delivered via the tail vein in a volume of 0.5 ml. Serum samples were collected once a week and total bilirubin (TB) levels were monitored. Untreated affected animals and wild-type or healthy littermates were used as controls.

Ugt1 mutant mice with a C57Bl / 6 background have been previously generated (Bortolussi et al., 2012). Wild-type litters were used as controls. Mice were bred and handled with full respect for EU Directive 2010/63 / EU for animal testing in accordance with institutional guidelines and experimental procedures approved by the Regional Institutional Review Board and relevant regulatory authorities. A gene mutation of the Ugt1a gene was introduced into an FVB / NJ mouse strain. The animals used in this study were at least 99.8% C57Bl / 6 mice or FVB / NJ genetic background obtained after more than 9 backcrosses with C57Bl / 6 mice and FVB / NJ, respectively. rice field. Mice were kept in a temperature controlled environment with a 12/12 hour light-dark cycle. The mice were free to receive standard solid feed and water. The vector was injected intraperitoneally on day 2 (P2) after birth and the bilirubin level was assayed 4 weeks after injection of the vector.

AAV dose:
The dose of vector administered is shown in the description of the figure.

Preparation of rat serum:
Blood samples were collected in a dry syringe once a week by puncturing the posterior orbital venous plexus. Blood was centrifuged at 4 ° C. at 8000 rpm, dispensed and frozen at −20 ° C.

Preparation of mouse plasma:
Blood samples were collected by cardiac puncture into a recovery syringe containing EDTA 4 weeks after injection into mutant and wild-type littermates. Blood was centrifuged at 2500 rpm, plasma was collected, dispensed and frozen at −80 ° C. All procedures were performed in a dimly lit place to avoid bilirubin degradation.

Measurement of rat bilirubin:
Measurements of total bilirubin in serum were performed using the bilirubin assay kit (Abnova, reference number KA1614) as described by the manufacturer. We performed the analysis using a volume of 50 μL of serum. Absorption values at 530 nm were obtained by using a multi-plate reader (PerkinElmer EnSpire).

Measurement of bilirubin in mice:
Measurements of total bilirubin in plasma were performed using the Direct and Total Bilirubin Reagent Kits (BQ Kits, San Diego, CA) with minor modifications as described by the manufacturer: scale of reaction. Was reduced and it was performed in a final volume of 300 μl (instead of 6000 μl) with only 10 μl plasma. Three commercially available bilirubin reference standards (control serum I, control serum II, and bilirubin calibration sample, Diazyme Laboratories, Poway, CA, USA) were included in the analysis of each set as quality controls. Absorption values at 560 nm were obtained by using a multi-plate reader (Perkin Elmer Envision Plate Reader, Walthman, MA, USA).

Western blot for liver extract:
Instantly frozen livers obtained from animals injected with any one of the three vectors were rapidly homogenized. Homogenates were used for the preparation of microsomes. The microsome extract was then placed on a Western blot, where the protein was detected using a polyclonal antibody against human UGT1. The protein band was quantified.

Results An optimized version of the codon of the human UGT1A1 coding sequence was generated and introduced into the expression plasmid. Two optimized UGT1A1 coding sequences (v2 and v3 sequences) and wild-type sequences were transfected into Huh-7 cells. The results obtained are reported in FIG. This experiment shows that the two codon-optimized sequences are translated more efficiently in vitro than wild-type sequences in human cells.

Panel A of FIG. 2 shows the levels of luciferase produced in Huh-7 cells by transfection with a plasmid expressing luciferase under transcriptional control of the hAAT promoter. Various intron sequences have been cloned into 5'of the luciferase coding sequence. Two of them, the HBB2 and FIX introns, were optimized by removal of ARF in the sequence performed by substituting one nucleotide in the ATG codon identified in the wild-type sequence of the intron. .. Expression of optimized constructs in hepatocyte lines indicates that removal of ARF from the intron sequence increases luciferase expression in both cases in vitro, indicating that the optimized HBB2 intron is particularly potent. Panel B compares the two plasmids and both express UGT1A1 under transcriptional control of the hAAT promoter. V2.0 contains a wild-type HBB2 intron, while v2.1 contains an optimized version. The data shown show that the v2.1 plasmid expresses 50% more UGT1A1 than v2.0 and there is no increase in mRNA levels.

Codon-optimized UGT1A1 version 2.0 and 2.1 AAV8 vectors (UGT1A1 2.0 and UGT1A1 2.1, respectively) were tested in vitro. The UGT1A1 2.0 vector and the UGT1A1 2.1 vector each contain a wild-type HBB2 intron (SEQ ID NO: 5) or a modified HBB2 intron (here ARF has been removed) (SEQ ID NO: 6). It depends only on the fact that it is. The results obtained are reported in FIG. This experiment shows that the codon-optimized UGT1A1 vector version 2.1 is more potent in vitro than the 2.0 version in human cells.

FIG. 4 shows the results of computerized analysis of the alternative reading frame (ARF) within the wild-type UGT1A1 vector (A) and the codon-optimized UGT1A1v2.1 vector (B). The v2.1 vector has a small number of ARFs compared to the wild-type sequence and is mostly opposite to the promoter. Furthermore, in FIG. 4, we have introduced ARF9 and 10 normally present in the HBB2 intron (used in the wild-type UGT1A1 construct presented in A) into the UGT1A1v2.1 optimized vector. It can be seen that it has been removed from the modified HBB2 intron of 6.

The codon-optimized AAV8-hAAT-coUGT1A1v2.1 vector was then administered at a dose of 5 × 10 ¹² vg / kg. Injection of the vector into the tail vein was performed in 6-week-old homozygous Gunn rats (UGT1A1-/-). In the graph of FIG. 5, injections of PBS into wild-type Gunn rats (WT, gray line), heterozygous Gunn rats (UGT1A1 +/-, dotted line), and homozygous Gunn rats (black line) are shown. Later, total bilirubin (TB) levels measured weekly are shown. All data are expressed as mean ± standard error. Injection of a codon-optimized vector completely corrected the disease phenotype.

The AAV8-hAAT-UGT1A1v2.1 vector was also administered at a dose of 5 × 10 ¹¹ vg / kg. The vector was administered by injection into the tail vein into 6-week-old homozygous Gunn rats (UGT1A1-/-). In the graph of FIG. 6, injections of PBS into wild-type Gunn rats (WT, gray line), heterozygous Gunn rats (UGT1A1 +/-, dotted line), and homozygous Gunn rats (black line) are shown. Later, total bilirubin (TB) levels measured weekly are shown. All data are expressed as mean ± standard error. Injection of a codon-optimized vector completely corrected the disease phenotype.

Two codon-optimized (v2.1 and v3) and wild-type AAV8-hAAT-UGT1A1 vectors were further administered at a dose of 5 × 10 ¹¹ vg / kg. Injection of the vector into the tail vein was performed in 6-week-old homozygous Gunn rats (UGT1A1-/-). The graph in FIG. 7A shows the level of total bilirubin (TB) measured weekly after injection. All data are expressed as mean ± standard error. As shown in Panel A of FIG. 7, injection of the three vectors completely corrected the disease phenotype. Two months after injection, animals were sacrificed and UGT1A1 protein levels were quantified by Western blot of liver homogenate. Panel B shows photographs of Western blots with antibodies specific for the UGT1A1 protein. Band quantification showed an increase in the amount of UGT1A1 protein in rats treated with AAV8-hAAT-coUGT1A1v2.1, even if the differences were not significant due to the large variability in expression levels observed in various animals. rice field.

Long-term efficacy was evaluated in 2-month-old Gunn rats injected with a 5 × 10 ¹² vg / kg AAV8-v2.1 UGT1A1 vector. Four months after injection, the average bilirubin level in blood was 1.75 mg / dL in male rats (initial level on day 0: 7.49, 77% decrease) and 0.85 mg / dL in female rats (initial level: 7.49, 77% decrease). Initial level on day 0: 6.15 mg / dL, 86% reduction). This result, showing a long-term modification of the phenotype, is particularly pronounced compared to a previous study by Pastore et al., Who reported a reduction of only 50% in bilirubin baseline levels in female rats using various constructs. .. In summary, the data presented show better in vivo efficacy compared to other vectors developed to cure CN by the process of the invention applied to AAV8-hAAT-coUGT1A1v2.1. It was shown that the vector having was brought.

We also tested the modifying efficacy of total bilirubin in a mouse model of Crigler-Najer syndrome. FIG. 8 is a graph showing total bilirubin (TB) levels one month after injection. Animals were injected with a dose of 3 × 10 ¹⁰ vg / mouse on the second day after birth (P2).

Untreated affected animals that remained alive with 15 days of phototherapy were used as controls (UNTR (PT)).

This experiment shows that the version 2.1 vector provides the highest level of total bilirubin modification of all vectors. All data are expressed as mean ± standard deviation. Each dot indicates an animal.

References

Claims (36)

Nucleic acid that is a codon-optimized UGT1A1 coding sequence in which the optimized coding sequence has an increased GC content compared to the wild-type coding sequence and / or a reduced number of alternative open reading frames. arrangement. The nucleic acid sequence of claim 1, comprising a nucleotide sequence that is at least 80% identical to the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3. The nucleic acid sequence of claim 2, comprising the nucleotide sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3. A nucleic acid construct comprising the nucleic acid sequence according to any one of claims 1 to 3. The nucleic acid construct according to claim 4, which is an expression cassette containing the nucleic acid sequence of any one of claims 1 to 3 operably linked to a promoter. The nucleic acid construct according to claim 5, wherein the promoter is a liver-specific promoter. The nucleic acid construct according to claim 5 or 6, wherein the promoter is selected from the group consisting of the hAAT promoter, the α-1 antitrypsin promoter (hAAT), the transthyretin promoter, the albumin promoter, and the thyroxine-binding globulin (TBG) promoter. The nucleic acid construct according to any one of claims 5 to 7, further comprising an intron. The nucleic acid construct of claim 8, wherein the intron is selected from the group consisting of human β-globin b2 (ie, HBB2) intron, FIX intron, and chicken β-globin intron. The nucleic acid construct of claim 8 or 9, wherein the intron is a modified intron with reduced ARF or no ARF at all. The nucleic acid construct of claim 10, wherein the modified intron is a modified HBB2 intron, a modified FIX intron, or a modified chicken β-globin intron. The modified intron described above may be the modified HBB2 intron set forth in SEQ ID NO: 6, the modified FIX intron set forth in SEQ ID NO: 8, or the modified chicken β-globin intron set forth in SEQ ID NO: 10. The nucleic acid construct according to claim 11. A vector containing the nucleic acid sequence or nucleic acid construct according to any one of claims 1 to 12. The vector according to claim 13, which is a viral vector. 13. The vector of claim 13, which is a retroviral vector, eg, a lentiviral vector, or an AAV vector. The vector according to claim 13 or 14, which is a single-stranded or double-stranded self-complementary AAV vector. The AAV vector has a capsid derived from AAV, such as AAV-1, -2, -5, -6, -7, -8, -9, -rh10, -rh74, -dj capsid, or a chimeric capsid. The vector according to claim 15 or 16. The vector according to any one of claims 15 to 17, wherein the AAV vector has an AAV8 capsid. The vector according to any one of claims 15 to 18, which is a pseudo-typed AAV vector. A cell transformed with the nucleic acid sequence of any one of claims 1 to 3, the nucleic acid construct of any one of claims 4 to 12, or the vector of any one of claims 13 to 19. The cell according to claim 20, which is a hepatocyte or a muscle cell. The nucleic acid sequence according to any one of claims 1 to 3, the nucleic acid construct according to any one of claims 4 to 12, for use in the treatment of Crigler-Najer syndrome type I or II or Gilbert's syndrome, claim. The vector according to any one of items 13 to 19, or the cell according to claim 20 or 21. An intron, a modified intron with a reduced open reading frame. Modified HBB2 introns, especially modified HBB2 introns of SEQ ID NO: 6, modified FIX introns, especially modified FIX introns of SEQ ID NO: 8, or modified chicken β-globin introns, especially modifications of SEQ ID NO: 10. The intron of claim 23, which is the chicken β-globin intron. A nucleic acid construct comprising the intron of claim 23 or 24. The nucleic acid construct of claim 24 further comprising the gene of interest and one or more additional expression control sequences, such as promoters and / or enhancers. The nucleic acid construct of claim 26, wherein the promoter is a ubiquitous or tissue-specific promoter, eg, a liver-specific promoter. A vector comprising the intron according to claim 23 or 24 or the nucleic acid construct according to any one of claims 25 to 27. 28. The vector of claim 28, which is a viral vector, such as a retroviral vector, particularly a lentiviral vector, or an AAV vector. The vector according to claim 28 or 29, which is a single-stranded or double-stranded self-complementary AAV vector. The AAV vector has an AAV-derived capsid, such as AAV-1, -2, -5, -6, -7, -8, -9, -rh10, -rh74, -dj capsid, or has a chimeric capsid. The vector according to claim 23 or 30. The vector according to any one of claims 29 to 31, wherein the AAV vector has an AAV8 capsid. The vector according to any one of claims 29 to 32, which is a pseudo-typed AAV vector. A cell transformed with the nucleic acid construct according to any one of claims 25 to 27 or the vector according to any one of claims 28 to 33. The cell according to claim 34, which is a hepatocyte or a muscle cell. The nucleic acid construct according to any one of claims 26 to 27, the vector according to any one of claims 28 to 33, wherein the gene of interest is a therapeutic gene for use in a method of gene therapy or cell therapy. , Or the cell according to any one of claims 34 to 35.

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